This invention relates to vaccines which protect against diphtheria toxin.
Wild-type diphtheria toxin (DT) is a protein exotoxin produced by the bacterium Corynebacteria diphtheria. The molecule is produced as a single polypeptide that is proteolytically cleaved at amino acid residue 190, 192, or 193 into two subunits linked by a disulfide bond: fragment A (N-terminal .about.21K) and fragment B (C-terminal .about.37K) (Moskaug, et al., Biol Chem 264:15709-15713, 1989; Collier et al., Biol Chem, 246:1496-1503, 1971). The receptor binding domain of wild-type DT is contained within the B fragment (Rolfe et al., J. Biol. Chem., 265:7331-7337, 1990). Fragment A is the catalytically active portion of wild-type DT. It is an NAD-dependent ADP-ribosyltransferase which inactivates protein synthesis factor elongation factor 2 (EF-2), thereby shutting down protein synthesis in the intoxicated cell. Fragment B of wild-type DT possesses the receptor-binding domain known as the R domain (amino acids 379-535, see Choe et al., Nature, 357:216-222, 1992; Fu et al., In Vaccines 93, Ginsberg et al., Eds., CSHSQB, pp. 379-383, 1993). The receptor-binding domain comprises 10 .beta. strands which form two .beta.sheets. A subset of the .beta.strands resembles an immunoglobulin-like moiety, which is conceivably involved in receptor recognition (Choe et al., Nature 357:216-222, 1992). Once DT is bound to the cell via the receptor binding domain, the receptor/DT complex is internalized. A second functional region on fragment B acts to translocate DT across the cell membrane, releasing catalytically active fragment A into the cytosol of the cell. A single molecule of fragment A is sufficient to inactivate cellular protein synthesis.
Immunity to a bacterial toxin such as wild-type DT may be acquired naturally during the course of infection, or artificially by injection of a detoxified form of the toxin (also called a chemical toxoid) (Germanier, ed., Bacterial Vaccines, Academic Press, Orlando, Fla., 1984). Chemical toxoids have traditionally been prepared by chemical modification of native toxins (e.g., with formalin or formaldehyde (Lingood et al., Brit. J. Exp. Path. 44:177, 1963), rendering them non-toxic while retaining antigenicity that protects the vaccinated animal. An example of a chemical toxoid is that described by Michel and Dirkx (Biochem. Biophys. Acta 491:286-295, 1977). However, a chemical toxoid may lose the added chemical group or groups, and revert to its active, toxic form, so that its use as a vaccine poses a risk to the vaccinee.
Another avenue for producing a toxoid is by the use of genetic techniques. A Corynebacterium diphtheriae mutant, CRM-197 (Uchida et al., J. Biol. Chem. 248:3838-3844, 1973; Uchida, et al., Nature 233:8-11, 1971) (CRM standing for "cross-reacting material") was shown to contain an enzymatically inactive DT protein which produces an anti-DT immune response. Collier et al. (U.S. Pat. No. 4,709,017; herein incorporated by reference) discloses a genetically engineered DT mutant that bears an amino acid deletion at Glu-148. Substitution of Asp, Gln or Ser at this site diminishes enzymatic and cytotoxic activities by 2-3 orders of magnitude, showing that the spatial location and chemical nature of the Glu-148 side chain greatly affects these activities (Carroll et al., J. Biol. Chem. 262:8707, 1987; Tweten et al., J. Biol. Chem. 260:10392, 1985; Douglas et al., J. Bacteriol. 169:4967, 1987).
Similarly, Greenfield et al. (U.S. Pat. No. 4,950,740; herein incorporated by reference) discloses genetically engineered mutant forms of DT in which the Glu 148 residue is deleted or replaced with Asn. The DNA sequence and corresponding amino acid sequence of naturally occurring diphtheria toxin DNA is set forth in FIG. 1 (SEQ ID NO:1).